Preparation of blood fraction for use in rh testing procedures



PREPARATION OF BLOOD FRACTION FOR USE m Rh TESTING PROCEDURES Alfred B. Kupferberg, Somerville, and Heron 0. Singher, Plainfield, N. J., assignors to Ortho Pharmaceutical Corporation, a corporation of New Jersey No Drawing. Application July 22, 1954, Serial No. 445,193

6 Claims. (Cl. 167-845) This invention relates to a process for the fractionation of mammalian serum and relates particularly to a process for preparing a serum fraction containing albumin in a major amount and globulin in a minor amount, said serum fraction being suitable for use in demonstrating the presence in blood of an Rh antibody known variously as incomplete antibody, blocking antibody, albumin agglutinin, and agglutinoid.

In 1939, Levine and Stetson, J. A. M. A., 113:126, 1939 reported a transfusion reaction in a woman who delivered a macerated fetus and whose serum contained agglutinins. in 1940, Landsteiner and Wiener, Proc. Exper. Biol. and Med. 43:223, 1940, reported the preparation of an antiserum against the red cells of the rhesus monkey and found that this anti-rhesus serum, when properly absorbed, agglutinated the blood of approximately 85% of the members of the white race that were tested. They labelled the antigen in the human bloods that reacted with the antirhesus serum the Rh factor. Human bloods containing the Rh factor were termed Rh-positive. it was shown that Levine and Stetson, who reported the earlier transfusion reaction, were dealing with a case of Rh immunization and that the unnamed property was the property later named the Rb factor by Landsteiner and Wiener. Levine, Katzin, and Burnham, I. A. M. A., 116:825, 1941, reported that intragroup incompatibility could be produced by immunization of an Rh-negative mother by an Rhpositive fetus. These investigators demonstrated at the same time that Rh immunization explained the majority of cases of erythroblastosis fetalis.

It was at first thought that the Rh types could be explained on the basis of the presence or absence of the Rb factor, a single positive factor. Later it was discovered that the problem was much more complex and at the present time at least eight Rh subtypes are demonstrable. The anti-Rh agglutinins are not found normally in the blood of any individual. Antibodies that agglutinate Rhpositive cells may develop in the serum of some Rhnegative individuals under certain conditions. Anti-Rh agglutinins, which appear in the blood of certain Rhnegative individuals as a result of immunization by transfusion of Rh-positive blood or by bearing Rh-positive children, are demonstrable in the serum or plasma of the immunized individual and are generally divided into two types; the saline agglutinins (complete antibodies) and albumin agglutinins (incomplete antibodies). Anti-Rh agglutinins in general are similar to other isohemagglutinins except that they are more active at 37 C. than at lower temperatures. The anti-Rh agglutinins act very Well when the serum is tested against a 1% or 2% suspension of Rh-positive cells in 0.85% sodium chloride. It was later discovered that there were antibodies other than the saline-active, anti-Rh agglutinins responsible for immun zation. It was noted that a large proportion of the Rh-negative women who bore erythroblastotic children did not show anti-Rh agglutinins in their serum when the serum was tested against a 1% or 2% suspension of Rhpositive cells in 0.85% sodium chloride. It was demonnited States Patent iution, and it was assumed that their serum possessed antibodies capable of nullifying the effect of ordinary anti- Rh agglutinins on Rh-positive cells suspended in saline. This antibody has been called incomplete antibody, blocking antibody, albumin agglutinin, and agglutinoid.

Hill, Reid, and Haberman, Texas State Journal of Medicine, 45:477481, 1949, concluded that the antibodyantigen reactions are reversible and that there is competition between the different varieties of antibodies which explains various antibody patterns observed clinically. These investigators employed the term agglutinoid to designate the R11 antibody found in the euglobulin associated with the albumin fraction which saturates the antigen and blocks the action of the saline agglutinin to distinguish it from the antibodies which these investigators have designated cryptagglutinoids which do not block, do not agglutinate saline suspensions, and show chemical differences to these two antibodies. These investigators postulate that the reaction of antibodies with antigen is reversible and that union of antigen and antibody occurs much more readily than disassociation. The three antibodies were found to vary in the aflinity for the antigen in the probable order agglutinoid (albumin agglutinins), agglutinin (saline agglutinins), and cryptagglutinoid. Reactions between antibodies and antigens follow the laws of mass action and therefore, are greatly influenced by the concentrations of any of the three orders of antibody that might be present in a given serum. Blocking effects as well as apparent masking of agglutinins are well explained by this concept of heterogeneous antibodies acting in reversible reactions with the antigen and following the laws of mass action. Fractionation studies of these investigators furnished further evidence for these three orders of Rh antibodies and it has also been demonstrated that there are significant chemical differences between the agglutinins (saline agglutinins), and agglutinoids (albumin agglutinins) and cryptagglutinoids.

A test for demonstrating the presence of the incomplete antibody was developed by Diamond and Abelson, J. Lab. and Clin. Med, 30:204, 1945, which demonstrated this antibody produced agglutination of Rh-positive cells. In the test, one drop of the serum containing the incomplete antibody was mixed with two drops of whole oxalated or citrated Rh-positive blood and spread thinly on a warm slide, and this resulted in clumping of the Rh-positive cells. The same results were obtained and could be demonstrated by testing the serum containing the incomplete antibody against a 50% suspension of Rh-positive cells suspended in serum. It was originally'thought that the heavy cell suspension was a factor which permitted such a serum to clump Rh-positive cells, but it was later concluded that the serum or plasma in Which the cells were suspended was the determining factor. It was still later shown that the same effect is possible when incomplete antibody is tested against a 2% suspension of Rh-positive cells suspended in a 20% to 30% solution of human or bovine albumin, acacia, globin and many other macromolecular substances. by incomplete antibodies waslatermodified and in this modification, the serum and Rh-positive cells in saline are incubated for one hourat 37 C. and centrifuged. The supernatant saline is removed and serum or other macromolecular substance addedfollowed by resuspension of the cells. The mixture isfagain centrifuged and observed for clumping. 'Inthis'te'st, the Rh=positive cells Patented Sept. 4,

The test to produce agglutination.

, saline absorb'the incomplete antibody but do not clump until the macromolecular solution is added.

E. A. McCulloch, Nature l65:276-277, 1950, reported Kekaviclr'and Mac'- plasma were inactive but that an active fraction could be 33.4%; beta-globulin, 127%; and gamma-globulin, 8.9%. Albumin; separated electrophoretically from this preparation and concentratedby freezeadrying showed no activity, but at a comparable protein ccncentra'tion, the

' globulins were'highly active.

these reported results that since the albumin was consistently negative, and no activity could be found in beta- McCulloch concluded from and gamma-globulin; the activity'appeared to be associated with alpha-globulin.'- This author suggested that alpha-globulin, is responsible for the demonstration of incomplete Rh antibodies'in the Diamond test.

Reid and Jones, Amer. J. Clin. Pathology, 19:10-15,

1949, described a processffor' the. production of thera peutic fractions of humanblood serum by the removal of salt with ion exchange resins. ,In the process of these investigators, cation-and anion exchange resins were em.- ployed alternately forremoving salts from blood serum or plasma, while keeping the pH betWeen 6 and 8. As the salt concentration progressively decreased, the glob- "ulins precipitated out until. the solution was essentially salt-free and at this time only euglobulins, albumin, hemoglobin, and other impurities were left in the solution.

Precipitated globulins were-removed: and the pH Was brought down to 5 which resulted in precipitation of v the 1 euglobulins; The euglobulin fraction contains the alphaglobulin components suggested by .Mcculloch to be necessary for demonstration of incomplete Rh antibodies I in the Diamond. test, as well as. beta-globulins.

iprecipitated .euglobulins were removed. and a solution of albumin containing thehemoglobin which was in the I original plasma remained. Sodium caprylate was added to 0.05 molarity of the albumin solution and the pH was The . 4 I solution, which. fraction contains albumin and alphaglobulins. J It has now been discoveredthat a serum fraction containing albumen and alpha-globulins which is suitable for use in the Diamond test maybe produced from mammalian blood serum by a- 'mtess in which salts are refprepared frornthe supernatant left after their precipitation which was soluble in 0.9%.saline at-pH 5.0. This soluble fraction 7 consisted .ofz-f albumin 38%; alpha-globulin,

moved fromihe-serum withion exchange resins to precipitate salt soluble-euglobulins and, after removing the precipitate,v globulinsin the serum are heat denatured in'the presence of a salt -ofan o'rgan'ic acidwithout-denaturation of albumin and alpha-globulins,:-and subsequently precipitated by an acida'djustment'of thepI-I of the solution and removed, following; which;..; the alpha-globulins :are precipitatedby pH adjustment, .the albumin is precipitated by'methanol addition and albumin and alpha-globulins are removed together by appropriate means such as centrifugation. V

lntheprocess of thisinvention, seriurn is passedthrough a resin column containingboth cation and. anion exchange resins in a proportionsuch thatasolution having'a pH within the rangefof from. .6 to; 8,.and preferably neutral, results on passage through the column of an acidic, basic, or neutral solution. I Cation. exchange resinshaving active phenolic, carboxyl or sulfonic groups which are saturated with hydrogen ions such as phenol methylene sulfonic acid resin, nuclear sulfonic acid resin, and polymerized acrylic-acid. resin andanion exchange resins having'active amine groups such as a polymer resulting from nitration and subsequent reduction ofa.'styrene-divinylbenzene copolymer are suitable for us e in theprocess of the'invention. As the resins remove the'salts from the serum, the

' salt-soluble globulins, also referred to as euglobulins, are

precipitated and subsequently removed by appropriate means such as centrifugation. 'An alkali .metal salt of a fatty acid having 4 to 10 carbon atomsyis then added to the solutionntoibring its molarity with respect thereto at least about 0.15. An alkalimetal saltof a fatty acid such as butyric, caproic andheXanoic acid and of acetyl tryptophane .has, beenfoundparticularly suitable for adjusted to 7.4. The caprylate stabilized the albumin so thatthe solution could be, pasteurized. The pH'of the solution was adjusted til-5.0 and this resulted in'denaturation of the hemoglobin which-wasthen quantitatively resolution was-dessicated under'vacuum from the frozen state and-by this means could be safely carried to any concentration. desired. The process of these investigatorsrproducedan;undenatured albuminsuitable for.

I clinical use and freefromglobulin fractions.

It is an object offthis invention to. prepare a serum 5 fraction suitable foriuse in demonstrating incomplete Rh antibodies a methoddescribed by :Diamond.

It is another object of this invention to prepare a serum fraction suitable for. use in demonstrating incomplete Rhantibodies by suspending the reacting cells in a con- 'centration of the serum fraction.

It is still another objectof this invention-to prepare a serum fraction, suitable for use inithe method of demonstrating incompletefRhantibodies ofDiamond in which the reacting- .cells are suspended in concentrated albumin addition to the solution. Belowabout 0.15 molar, heat subsequently applied tothe solution destroys active alphaglobulin components necessary 'f 0l the test; when the molarity is greater than about 0.3, .alpha-globulins are precipitated. ThepH. is .-then.adjusted.- to between 6.8

and 7.4, but it is preferredlthatthe pH be at 7.0. The

solution is brought to atemperature of approximately 75 C. and maintained at this temperature for about 25 to :35 minutes. .Thealkali metalsalt prevents denaturation by heating of .the alpha-globulins and albumin present in the. solution. Heating to this temperature substantially denatures globulinsf present in the solution other than .the activeportions of the alphmglobulins. The solutionisthen cooledItoa temperature not greater .than about 25 C..and.adjusted..to .a.pH.of.5.5 to 5.8, the preferred pH beingS eL 'Adjustmentof .the pH of the solution to 5.5 .to 5.8.afterheatingresults in precipitation of substantially all denatured globulins. leaving undenat ured alpha-globulins .in. solution. If the pH is adjusted :to a more acid pH than 5.5,. alpha-globulins are precipitated and if the pH is adjusted toa more alkalinereaction there is insufficient precipitation of denatured globulins. The precipitated denatured. globulins are removed by appropriate means suchas centrifugation' and the pH of the supernatant is adjusted .to within the range of from 3.0 to 5.5. Uponzadjustment ofi the pHof the supernatant to a point within this range, alpha-globulins are precipitated. It is preferred...that the pH be adjusted to a point within the rangeof 4.4 .to 4.8 becausethis is the point of minimum solubility of albumin in water and is:known as the isoelectric range. If'thepH of the supernatant is adjusted to apoint below 3.0, albumin'is destroyed and if the pHisadjus'tedtoa point: above 5. 5, alpha-globulins are not precipitated, After adjustment of the pH, the supernatantis cooledlto a temperature within the range of from 2 to 1 C. andzmethanol in an amount .to bring the quantity thereof to at least 38% by volume is added to the supernatant at such rate that the temperature of the solution does not rise above 2 C. The addition may be facilitated if the methanol is chilled before addition to the supernatant. It is essential that alpha-globulins be precipitated before methanol is added to the supernatant and that methanol not be in contact with the alphaglobulins and albumin at a temperature above 2 C. because methanol will denature any alpha-globulins in solution and denaturation of both alpha-globulins and albumin will occur at a higher temperature. The methanol precipitates the albumin fraction from the solution and precipitated albumin and alpha-globulins are removed by centrifugation. Residual alcohol present in the precipitate is removed by evaporation, preferably under vacuum at a temperature lower than 2 C. The residual solid material may be redissolved in an appropriate amount of a solution of 0.9% aqueous sodium chloride and adjusted to a pH of from 7.0 to 7.4 to give a final solution with a concentration of from 26 to 30% protein. This is the concentration and pH range of the protein fraction used in the Diamond test. The invention is further illustrated by the following example, but it should be understood that the invention is not limited to the specific details set forth in the examples.

50 liters of bovine serum were passed through a resin column consisting of a mixture of a strong-base anion exchange resin, Amberlite IRA410 having a total capacity of 2.5 milliequivalents per gram, the resin being a reaction product of trimethyl amine and an insoluble, crosslinked copolymer of styrene and divinyl benzene, which copolymer contains chloroaLkyl groups having the formula CnHZnCl, in which CnHzn is an alkylene group and in which n is an integer from 1 to 4, and a phenol methylene sulfonic cation exchange resin, Amberlite IR-105G. The proportion of the cation exchange resin and anion exchange resin in the column was such that a neutral solution resulted on passage through the column of an acidic, basic, or neutral solution. The eflluent from the column contained precipitated globulin which was removed from the solution by centrifngation. The total solution of the eflluent was 60 liters and after precipitated globulins had been removed therefrom, 1467 grams of sodium caprylate were added to the supernatant. The pH was adjusted to 7.0 and the solution was heated at 75 C. for thirty minutes, cooled to room temperature and the pH was then adjusted to 5.6 at which time a precipitation of denatured globulins resulted which was removed by centrifugation. The supernatant having a volume of 40 liters was adjusted to a pH of 4.5 and cooled to C. 26.6 liters of chilled methanol were added slowly and at such a rate that the temperature of the solution was maintained approximately at 0 C. throughout the addition. The precipitate resulting from the addition of methanol was removed by centrifugation and residual alcohol present in the removed precipitate was evaporated under vacuum at -5 C. The precipitate was dissolved in 3.5 liters of 0.9% aqueous sodium chloride and the pH was adjusted to 7.2. 4.5 liters of 28.25% protein fraction containing bovine albumin and alpha-globulins were obtained.

The following example illustrates the use of the solution of protein fraction obtained by the above procedure in the test procedure of Diamond, the said solution of protein fraction being substituted for the solution of human or bovine albumin, acacia, globin or other macromolecular substance used in the Diamond procedure.

A blood sample suspected of containing the blocking antibody was allowed to clot and the clotted sample was centrifuged. Two drops of the serum obtained by centrifugation were placed in a test tube and to this was added one drop of a physiological salt solution containing in solution 22% by weight of the blood fraction prepared according to the example above, and 2% by weight of Rh positive blood cells suspended therein. The mixture was incubated for one hour in a water bath maintained at 37 C., removed from the water bath; centrifuged at 1000 revolutions per minute for one minute, removed from the centrifuge, agitated gently and examined microscopically. The red blood cells exhibited the phenomenon known as clumping, thus demonstrating the presence in the test serum of the blocking antibody.

Since certain changes may be made in the above process and different embodiments of the invention could be made without departing from its scope, it is intended that all matter contained in the above description should be interpreted as illustrative and not in a limiting sense.

This application is a continuation in part of our application Serial No. 273,781, filed February 27, 1952.

What is claimed is:

1. The process of preparing blood serum proteins suitable for use in demonstrating incomplete Rh antibodies by suspending reacting blood cells in a solution of the blood serum proteins which comprises passing mammalian blood serum through a column containing both cation and anion exchange resins in a proportion such that a solution having a pH within the range of from 6 to 8 results on passage through the column of a solution selected from the class consisting of acidic, basic, and neutral solutions, whereby a precipitate of euglobulins is formed in the solution; removing the precipitate, adding to the solution an alkali metal salt of an organic acid selected from the group consisting of fatty acids having from 4 to 10 carbon atoms and acetyltryptophane in an amount sufficient to bring the molarity of the solution with respect thereto to at least about 0.15; adjusting the pH of the solution to 6.8-7.4; heating the solution to approximately 75 C. and maintaining the temperature of the solution at approximately this temperature for a period of from 25 to 35 minutes; cooling the solution to a temperature not greater than about 25 C.; adjusting the pH of the solution to 5.5-5.8; whereby a precipitate of denatured globulins is formed; removing the precipitated denatured globulins from the solution; adjusting the pH of the solution to 3.0-5.5, whereby alpha-globulins are precipitated, cooling the solution to a temperature of 2 to -1 C. and adding methanol to the solution in an amount sufiicient to bring the amount with respect thereto to at least 38% by volume and at such a rate that the temperature of the solution does not rise above 2 C. during the course of the addition, whereby albumin is precipitated; and removing precipitated alpha-globulins and albumin from the solution.

2. A process according to claim 1 in which the alkali metal salt of an organic acid is sodium caprylate.

3. A process according to claim 1 in which the cation exchange resin has an active group selected from the class consisting of phenolic, carboxyl, and sulfonic groups and in which the anion exchange resin has an active amine group.

4. A process according to claim 1 in which the cation exchange resin is a phenol methylene sulfonic acid resin and in which the anion exchange resin is a polymer resulating from nitration and subsequent reduction of a styrene-divinylbenzene copolymer.

5. The process of preparing bovine blood serum proteins suitable for use in demonstrating incomplete Rh antibodies by suspending reacting blood cells in a solution of the blood serum proteins which comprises passing bovine blood serum through a column containing both cation and anion exchange resins in a proportion such that a solution having a pH within the range of from 6 to 8 results on passage through the column of a solution selected from the class consisting of acidic, basic, and neutral solutions, whereby a precipitate of euglobulins is formed in the solution; removing the precipitate, adding to the solution an alkali metal salt of an organic acid selected from the group consisting of fatty acids having from 4 to 10 carbon atoms and acetyltryptophane in an amount sufficient to bring the molarity of the solution with respect thereto to at least about 0.15; adjusting the pH of the solution to 6.8-7.4; heating the solution to approximately 75 C. and maintaining .the temperature.

of the .solution at approximately :this temperaturefor .a period of. from 25 to 35'minutes; cooling .the solutionio a temperature not .greater than about 25 C.; adjusting the pH of the solution to 5.5-5.8; whereby a precipitate of denatured globulins is formed;.removingtheprecipitated denatured .globulinsfrom the solution; adjusting the .pH of the solution to 3.0-5.5, whereby alphaglobulinsare precipitated, cooling .thesolution to a .tem-

perature .of 2. to 1 C. and adding methanol to the,

solution in an amount suflicient to bring .the amount with respect thereto to at least 38% by volume and at such a rate that the temperature .of the solution does not rise above 2 C. during the course of the addition, whereby albumin is precipitated; and removing precipitatedalphaglobulins and albumin from the solution.

"6. A processaccording .toclairn Sin whichlthe .alkali metal saltof sanorganicracidis sodium :caprylate.

:References Citedin the file of this patent UNITED STATES PATENTS Williamset a1. Mar. 2, 1948 OTHER REFERENCES Liu 'et 211.: Chinese J. Physiol., vol. 8, 'No. 2, 1934, pp. 97-109 (pp. 97, 98, 99, 100, 101 and 109 relied upon).

McCulloch: Nature, vol. 165, pp. 276-277, Feb. 18,. i 

1. THE PROCESS OF PREPARING BLOOD SERUM PROTEINS SUITABLE FOR USE IN DEMOSTRATING INCOMPLETE RH ANTIBODIES BY SUSPENDING REACTING BLOOD CELLS IN A SOLUTION OF THE BLOOD SERUM PROTEINS WHICH COMPRISES PASSING MAMMALIAN BLOOD SERUM THROUGH A COLUMN CONTAINING BOTH CATION AND ANION EXCHANGE RESINS IN A PROPORTION SUCH THAT A SOLUTION HAVING A PH WITHIN THE RANGE OF FROM 6 TO 8 RESULTS ON PASSAGE THROUGH THE COLUMN OF A SOLUTION SELECTED FROM THE CLASS CONSISTING OF ACIDIC, BASIC, AND NEUTRAL SOLUTIONS, WHEREBY A PRECIPITATE OF EUGLOBULINS IS FORMED IN THE SOLUTION; REMOVING THE PRECIPITATE, ADDING TO THE SOLUTION AN ALKALI METAL SALT OF AN ORGANIC ACID SELECTED FROM THE GROUP CONSISTING OF FATTY ACIDS HAVING FROM 4 TO 10 CARBON ATOMS AND ACETYLTRYPTOPHANE IN AN AMOUNT SUFFICIENT TO BRING THE MOLARITY OF THE SOLUTION WITH RESPECT THERETO TO AT LEAST ABOUT 0.15; ADJUSTING THE PH OF THE RESOLUTION TO 6.8-7.4; HEATING THE SOLUTION TO APPROXIMATELY 75* C. AND MAINTAINING THE TEMPERATURE OF THE SOLUTION A T APPROXIMATELY THIS TEMPERATURE FOR A PERIOD OF FROM 25 TO 35 MINUTES; COOLING THE SOLUTION TO A TEMPERATURE NOT GREATER THAN ABOUT 25* C.; ADJUSTING THE PH OF THE SOLUTION TO 5.5-5.8; WHEREBY A PRECIPITATE OF DENATURED GLOBULINS IS FORMED; REMOVING THE PRECIPITATED DENATURED GLOBULINS FROM THE SOLUTION; ADJUSTING THE PH OF THE SOLUTION TO 3.0-5.5, WHEREBY ALPHA-GLOBULINS ARE PRECIPITAED, COOLING THE SOLUTION TO A TEMPERATURE OF 2* TO - 1* C. AND ADDING METHANOL TO THE SOLUTION IN AN AMOUNT SUFFICIENT TO BRING THE AMOUNT WITH RESPECT THERETO TO AT LEAST 38% BY VOLUME AND AT SUCH A RATE THAT THE TEMPERATURE OF THE SOLUTION DOES NOT RISE ABOVE 2* C. DURING THE COURSE OF THE ADDITION, WHEREBY ALBUMIN IS PRECIPITATED; AND REMOVING PRECIPITATED ALPHA-GLOBULINS AND ALBUMIN FROM THE SOLUTION. 